PROJECT SUMMARY This proposal aims to define the poorly understood yet critical relationship between human NK cell migration and differentiation. By integrating our validated system of human NK cell differentiation and highly quantitative imaging and image analysis, we will 1) define the requirement for migration in human NK cell differentiation, and 2) dissect the molecular interactions between NK cell developmental intermediates and stromal cells. Natural killer (NK) cells are required for our body's defense against viral infection and malignancy; they also shape the outcome and success of transplantation. Despite their documented requirement in human health, NK cell development and acquisition of function is poorly understood. Interactions with accessory cells such as lymphoid stromal cells are required for human NK cell development, yet the molecular biology behind these cell-cell interactions is not known and has never been visualized. Using in vitro differentiation of human NK cells and quantitative imaging, we have described the contacts formed between developing NK cells and stromal cells, which we termed the developmental synapse. In addition, we have defined distinct migratory phenotypes associated with the progressive maturation of human NK cells on stromal cells. To dissect the relationship between NK cell development and migration on stromal cells, we will pursue the following specific aims: 1) determine the requirement for migration on stroma in human NK cell development; using both mechanical restraint and NK precursors from patients with rare mutations that impair NK cell migration, we will quantitatively measure the effect of inhibiting cell migration on human NK cell differentiation. To further delineate the role that adhesion and migration plays in human NK cell development, we will: 2) define the physical contribution of stromal cells to NK cell development. This will include the evaluation alternative substrates to determine the contribution of adhesiveness and substrate stiffness on NK cell development. Finally, we will 3) define the structural and functional relationship between the NK cell uropod and developmental synapse. This will delineate the mechanism of signal transduction that occurs in cells conjugated through the developmental synapse and define how this is related to the uropod formed in migrating cells. This final aim will be performed in situ and in vitro to correlate the physiological developmental synapse with that formed in culture. Through these aims we will advance our knowledge of the contact-dependent requirements for human NK cell development, a field in which few scientific advances have been made in the past ten years. Our ultimate goal is to identify necessary and sufficient mechanical and biochemical signals required to recapitulate human NK cell development to efficiently generate therapy cells without the use of stromal cell feeders. By understanding the requirement for cell migration in this process, we will generate findings of both clinical and scientific impact.